Proteomics Informatics Databases, data repositories and - - PowerPoint PPT Presentation

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Proteomics Informatics Databases, data repositories and - - PowerPoint PPT Presentation

Aug 13, 2023 230 likes •854 views

Proteomics Informatics Databases, data repositories and standardization (Week 7) Protein Sequence Databases RefSeq Distinguishing Features of the RefSeq collection include: non-redundancy explicitly linked nucleotide and protein

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Proteomics Informatics – Databases, data repositories and standardization (Week 7)

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Protein Sequence Databases

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RefSeq

http://www.ncbi.nlm.nih.gov/books/NBK21091/

Distinguishing Features of the RefSeq collection include:

  • non-redundancy
  • explicitly linked nucleotide and protein sequences
  • updates to reflect current knowledge of sequence data and biology
  • data validation and format consistency
  • ongoing curation by NCBI staff and collaborators, with reviewed

records indicated

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Ensembl

http://www.ensembl.org/

  • genome information for sequenced chordate genomes.
  • evidenced-based gene sets for all supported species
  • large-scale whole genome multiple species alignments across vertebrates
  • variation data resources for 17 species and regulation annotations based
  • n ENCODE and other data sets.
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UniProt

http://www.uniprot.org/

The mission of UniProt is to provide the scientific community with a comprehensive, high-quality and freely accessible resource of protein sequence and functional information.

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Species-Centric Consortia

For some organisms, there are consortia that provide high-quality databases:

Yeast (http://yeastgenome.org/) Fly (http://flybase.org/) Arabidopsis (http://arabidopsis.org/)

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FASTA

http://en.wikipedia.org/wiki/FASTA_format

RefSeq: >gi|168693669|ref|NP_001108231.1| zinc finger protein 683 [Homo sapiens]

MKEESAAQLGCCHRPMALGGTGGSLSPSLDFQLFRGDQVFSACRPLPDMVDAHGPSCASWLCPLPLAPGRSALLACLQDL DLNLCTPQPAPLGTDLQGLQEDALSMKHEPPGLQASSTDDKKFTVKYPQNKDKLGKQPERAGEGAPCPAFSSHNSSSPPP LQNRKSPSPLAFCPCPPVNSISKELPFLLHAFYPGYPLLLPPPHLFTYGALPSDQCPHLLMLPQDPSYPTMAMPSLLMMV NELGHPSARWETLLPYPGAFQASGQALPSQARNPGAGAAPTDSPGLERGGMASPAKRVPLSSQTGTAALPYPLKKKNGKI LYECNICGKSFGQLSNLKVHLRVHSGERPFQCALCQKSFTQLAHLQKHHLVHTGERPHKCSVCHKRFSSSSNLKTHLRLH SGARPFQCSVCRSRFTQHIHLKLHHRLHAPQPCGLVHTQLPLASLACLAQWHQGALDLMAVASEKHMGYDIDEVKVSSTS QGKARAVSLSSAGTPLVMGQDQNN

Ensembl: >ENSMUSP00000131420 pep:known supercontig:NCBIM37:NT_166407:104574:105272 gene:ENSMUSG00000092057 transcript:ENSMUST00000167991

MFSLMKKRRRKSSSNTLRNIVGCRISHCWKEGNEPVTQWKAIVLGQLPTNPSLYLVKYDGIDSIYGQELYSDDRILNLKVL PPIVVFPQVRDAHLARALVGRAVQQKFERKDGSEVNWRGVVLAQVPIMKDLFYITYKKDPALYAYQLLDDYKEGNLHMIPD TPPAEERSGGDSDVLIGNWVQYTRKDGSKKFGKVVYQVLDNPSVFFIKFHGDIHIYVYTMVPKILEVEKS

UniProt: >sp|Q16695|H31T_HUMAN Histone H3.1t OS=Homo sapiens GN=HIST3H3 PE=1 SV=3

MARTKQTARKSTGGKAPRKQLATKVARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLMREIAQDFK TDLRFQSSAVMALQEACESYLVGLFEDTNLCVIHAKRVTIMPKDIQLARRIRGERA

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PEFF - PSI Extended Fasta Format

>sp:P06748 \ID=NPM_HUMAN \Pname=(Nucleophosmin) (NPM) (Nucleolar phosphoprotein B23) (Numatrin) (Nucleolar protein NO38) \NcbiTaxId=9606 \ModRes=(125|MOD:00046)(199|MOD:00047) \Length=294 >sp:P00761 \ID=TRYP_PIG \Pname=(Trypsin precursor) (EC 3.4.21.4) \NcbiTaxId=9823 \Variant=(20|20|V) \Processed=(1|8|PROPEP)(9|231|CHAIN) \Length=231

http://www.psidev.info/node/363

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Sample-specific protein sequence databases

Protein DB

Identified and quantified peptides and proteins MS Samples Peptides

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Sample-specific protein sequence databases

Next-generation sequencing

  • f the genome

and transcriptome

Sample-specific Protein DB

Identified and quantified peptides and proteins MS Samples Peptides

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Sample-specific protein sequence databases

Next-generation sequencing

  • f the genome

and transcriptome

Sample-specific Protein DB

Identified and quantified peptides and proteins MS Samples

Exon 1 Somatic and germ-line mutations

TCGAGAGCTG TCGAGAGCTG TCGAGAGCTG TCGAGAGCTG TCGAGAGCTG TCGATAGCTG

Exon 1 Exon 2 Exon 3 Alternative Splicing

Novel Expression Exon 1 Exon 2

Gene X Exon 1 Gene X Exon 2 Gene Y Exon 1 Gene Fusions Gene Y Exon 2

Peptides

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Proteomics and Transcriptomics

  • f Breast Tumors
  • --250,000
  • --75,000
  • --50,000
  • --37,000
  • --10,000
  • --15,000
  • --20,000
  • --25,000
  • --100,000
  • --150,000

1 2 3 4 5 6 7 8

Sample Loading Chamber Sample Collection Chamber Resolving Gel Stacking Gel ABI 5600 Triple TOF

Primary Breast tumor Xenograft tumor

Illumina HiSeq

RNA-Seq MS/MS

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0.00001 0.0001 0.001 0.01 0.1 1 5 10 15

Frequency Number of amino acid changes

Basal germline Basal somatic Luminal germline Luminal somatic

Germline and Somatic Variants

The frequency of proteins as a function of the number of amino acid changes due to germline and somatic variants for the basal and luminal breast tumor xenografts

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Alternative Splicing

The number of exon/exon junctions as a function of the number

  • f RNA-Seq reads for the basal breast tumor xenograft.

1 10 100 1000 10000 100000 1 10 100 1000 10000

Number of junctions Number of reads

Model Exons Alternative splicing Novel expression

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Protein identification using sample-specific sequence databases

Protein DB + germline / somatic variants Tumor genome sequence 362 9 Germline variants Somatic variants Potentially novel peptides Tumor RNA-Seq

1114

Potentially novel peptides Spans splice site

70

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Data Repositories

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ProteomeExchange

http://www.proteomeexchange.org/

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PRIDE

http://www.ebi.ac.uk/pride/

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PeptideAtlas

http://www.peptideatlas.org/

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The Global Proteome Machine Databases (GPMDB)

http://gpmdb.thegpm.org

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Comparison with GPMDB

Most proteins show very reproducible peptide patterns

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Comparison with GPMDB

Query Spectrum Best match In GPMDB Second best match In GPMDB

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GPMDB usage last month

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GPMDB usage last month

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GPMDB Data Crowdsourcing

Any lab performs experiments Raw data sent to public repository (TRANCHE, PRIDE) Data imported by GPMDB Data analyzed & accepted/rejected General community uses information and inspects data Accepted information loaded into public collection

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Information for including a data set in GPMDB

  • 1. MS/MS data (required)
  • 1. MS raw data files
  • 2. ASCII files: mzXML, mzML, MGF, DTA,

etc.

  • 3. Analysis files: DAT, MSF, BIOML
  • 2. Sample Information (supply if possible)
  • 1. Species : human, yeast
  • 2. Cell/tissue type & subcellular localization
  • 3. Reagents: urea, formic acid, etc.
  • 4. Quantitation: SILAC, iTRAQ
  • 5. Proteolysis agent: trypsin, Lys-C
  • 3. Project information (suggested)
  • 1. Project name
  • 2. Contact information
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How to characterize the evidence in GPMDB for a protein? High confidence Medium confidence Low confidence No observation

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Star t End N

  • 2
  • 3
  • 4
  • 5
  • 6
  • 7
  • 8
  • 9
  • 10
  • 11 Skew Kurt

214 248 539 0.15 0.18 0.22 0.17 0.15 0.07 0.03 0.01 0.01 0.00 -0.01 -2.01 249 267 1010 0.04 0.09 0.13 0.16 0.16 0.14 0.13 0.06 0.04 0.05 -0.08 -1.89 182 196 832 0.09 0.15 0.20 0.19 0.18 0.13 0.05 0.01 0.00 0.00 -0.12 -1.84 250 267 4 0.25 0.00 0.25 0.00 0.25 0.00 0.00 0.00 0.00 0.25 0.48 -2.28 1 24 269 0.10 0.12 0.12 0.17 0.12 0.12 0.14 0.04 0.04 0.03 -0.33 -0.88 24 65 51 0.22 0.22 0.20 0.14 0.06 0.00 0.04 0.08 0.02 0.04 0.47 -1.62 66 101 334 0.09 0.08 0.11 0.11 0.09 0.11 0.09 0.13 0.08 0.12 0.10 -1.21 249 273 60 0.02 0.00 0.20 0.10 0.13 0.25 0.20 0.07 0.03 0.00 0.45 -1.36 214 242 10 0.00 0.10 0.00 0.00 0.00 0.00 0.30 0.20 0.20 0.20 0.54 -1.39 214 239 32 0.03 0.06 0.16 0.16 0.09 0.22 0.09 0.16 0.00 0.03 0.20 -0.99 111 120 117 0.09 0.20 0.15 0.26 0.29 0.01 0.00 0.00 0.00 0.00 0.62 -1.36 251 267 16 0.00 0.00 0.13 0.25 0.19 0.13 0.13 0.13 0.06 0.00 0.24 -0.60 214 241 14 0.00 0.00 0.00 0.07 0.29 0.21 0.07 0.29 0.00 0.07 0.87 -0.97 159 174 100 0.30 0.25 0.31 0.03 0.07 0.03 0.01 0.00 0.00 0.00 0.99 -1.07 68 101 10 0.00 0.00 0.00 0.00 0.00 0.20 0.10 0.10 0.30 0.30 0.86 -0.91 235 248 30 0.00 0.03 0.00 0.00 0.30 0.20 0.23 0.13 0.03 0.07 0.81 -0.82

Statistical model for 212 observations of TP53

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Statistical model for observations of DNAH2

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Statistical model for observations of GRAP2

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DNA Repair

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DNA Repair

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TP53BP1:p, tumor protein p53 binding protein 1

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TP53BP1:p, tumor protein p53 binding protein 1

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Sequence Annotations

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TP53BP1:p, tumor protein p53 binding protein 1

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TP53BP1:p, tumor protein p53 binding protein 1

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Peptide observations, catalase

Peptide Sequence Observations FSTVAGESGSADTVR 2633 FNTANDDNVTQVR 2432 AFYVNVLNEEQR 1722 LVNANGEAVYCK 1701 GPLLVQDVVFTDEMAHFDR 1637 LSQEDPDYGIR 1560 LFAYPDTHR 1499 NLSVEDAAR 1400 FYTEDGNWDLVGNNTPIFFIR 1386 ADVLTTGAGNPVGDK 1338

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Peptide Sequence

ω

FSTVAGESGSADTVR 0.08 FNTANDDNVTQVR 0.07 AFYVNVLNEEQR 0.05 LVNANGEAVYCK 0.05 GPLLVQDVVFTDEMAHFDR 0.05 LSQEDPDYGIR 0.04 LFAYPDTHR 0.04 NLSVEDAAR 0.04 FYTEDGNWDLVGNNTPIFFIR 0.04 ADVLTTGAGNPVGDK 0.04

Peptide frequency (ω), catalase

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0.00 0.02 0.04 0.06 0.08 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 ω

Peptide sequences

Global frequency of observation (ω), catalase

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For any set peptides observed in an experiment assigned to a particular protein (1 to j ):

∑

= Ω

j j

protein ω ) ( 1 ) ( ≤ Ω protein

Omega (Ω) value for a protein identification

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Protein ID

Ω (z= 2) Ω (z= 3)

SERPINB1 0.88 0.82 SNRPD1 0.88 0.59 CFL1 0.81 0.87 SNRPE 0.8 0.81 PPIA 0.79 0.64 CSTA 0.79 0.36 PFN1 0.76 0.61 CAT 0.71 0.78 GLRX 0.66 0.8 CALM1 0.62 0.76 FABP5 0.57 0.17

Protein Ω’s for a set of identifications

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Retention Time Distribution

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Mass Accuracy

0.05 0.1 0.15 0.2 0.25

  • 5

5 10 15 20 Mass Error [ppm]

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GO Cellular Processes

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KEGG Pathways

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Open-Source Resources

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ProteoWizard

http://proteowizard.sourceforge.net

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Protein Prospector

http://prospector.ucsf.edu/

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PROWL

http://prowl.rockefeller.edu/

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Proteogenomics - PGx

http://pgx.fenyolab.org/

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UCSC Genome Browser

http://genome.ucsc.edu/

RNA-Seq: Expression RNA-Seq: coverage Global PNNL Global WashU Phospho PNNL Somatic Variants Germline Variants RefSeq Genes

  • Alt. Splicing

Junctions Global Pep PNNL Phospho PNNL Global Pep WashU

1 2 3 4 5 6 7 8 9 10 11 12 13

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Slice - Scalable Data Sharing for Remote Mass Informatics

Most mass spectrometry data is acquired in discovery mode, meaning that the data is amenable to open-ended analysis as our understanding of the target biochemistry increases. In this sense, mass spectrometry based discovery work is more akin to an astronomical survey, where the full list

  • f object-types being imaged has not yet been fully elucidated, as opposed

to e.g. micro-array work, where the list of probes spotted onto the slide is finite and well understood.

slice.ionomix.com

Developed by Manor Askenazi

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Standardization

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Standardization - MIAPE

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Standardization – MIAPE-MSI

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Standardization – XML Formats

mzML - experimental results obtained by mass spectrometric analysis of biomolecular compounds mzIdentML - describe the outputs of proteomics search engines TraML - exchange and transmission of transition lists for selected reaction monitoring (SRM) experiments mzQuantML - describe the outputs of quantitation software for proteomics mzTab - defines a tab delimited text file format to report proteomics and metabolomics results. MIF - decribes the molecular interaction data exchange format. GelML - describes the processing and separations of proteins in samples using gel electrophoresis, within a proteomics experiment.

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Standardization - mzML

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Standardization - mzIdentML

MzIdentML CvList AnalysisSoftwareList AnalysisSampleCollection SequenceCollection AnalysisCollection AnalysisProtocolCollection DataCollection

URLs of controlled vocabularies used within the file Software packages used Biological samples analysed, annotated with CV terms Database entries of protein / peptide sequences identified and modifications Application of protocol inputs = external spectra1..n

  • utput = SpectrumIdentificationList 1

SpectrumIdentificationProtocol ProteinDetectionProtocol

SpectrumIdentificationProtocol AdditionalSearchParams ModificationParams Enzymes DatabaseFilters Parameters for the protein detection procedure

Inputs AnalysisData

AnalysisData SpectrumIdentificationList The database searched and the input file converted to mzIdentML

SpectrumIdentificationResult SpectrumIdentificationItem

ProteinDetectionList

ProteinAmbiguityGroup ProteinDetectionHypothesis

All identifications made from searching one spectrum One (poly)peptide- spectrum match A set of related protein identifications e.g. conflicting peptide-protein assignments A single protein identification

SpectrumIdentification ProteinDetection

Application of protocol inputs = SpectrumIdentificationList 1..n

  • utput = ProteinDetectionList1
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Proteomics Informatics – Databases, data repositories and standardization (Week 7)